Endocrine therapy results in a major survival benefit for women with breast cancers that overexpress estrogen (ERa) and progesterone (PR, A+B isoforms) receptors, while the absence of PR due to an activated but potentially defective tumor ERalpha mechanism (ER+/PR- phenotype) identifies those with early or advanced disease less likely to benefit from endocrine therapy. This project first demonstrated that oxidant stresses can induce both thiol reversible and irreversible structural modifications within the ERalpha DNA-binding domain (ER-DBD), impairing ER DNA-binding and altering ER-inducible gene expression. Such structurally modified receptors may be found in up to a third of all ER-positive breast tumors, in part accounting for their inability to co-express ER-inducible genes like PR or respond to endocrine therapy. Evaluation of oxidant stress effects on ERalpha by mass spectrometry (MS) has defined a portion of one Zn finger (ZnF2B) within the ER-DBD most sensitive to oxidant stress, enabling production of a first-generation antibody that can specifically recognize oxidant modified ERalpha (oxER) as found in some breast tumor extracts. A new cell culture model replicating additional aspects of oxidant stressed ERa as found in human breast tumors has demonstrated that in a dose-dependent fashion redox-active and arylating quinones like menadione/vitamin K3 produce thiol irreversible loss of ER-DNA binding, Ser-118 phosphorylation of full-length (67 kDa) and truncated (45-55 kDa) variants of nuclear ERa and proteasomal decay of ERalpha. This project will now pursue two complementary aims: 1) further MS characterization of endogenously produced oxER and development of newer generation antibodies capable of specifically detecting other forms of oxER; and 2) continued assessment of the functional consequences of oxidant stressed ERalpha in model cell systems treated with different chemical stresses, and clinical evaluation of oxER-associated parameters measured in two different study samples of ER-positive breast tumors, one dichotomized by PR status and the other by age at diagnosis. The ERalpha mechanistic defects being measured and associated with oxER formation include loss of intracellular ERalpha DNA-binding, formation of phosphorylated and truncated ERalpha isoforms, altered endogenous PR isoforms and ER-associated gene expression signatures. Together, these aims will relate oxidant stress responses with specific oxER-associated mechanistic pathways, and validate the clinical significance of measuring oxER-associated parameters in human breast tumors.